Mitochondrial alpha-keto acid dehydrogenase kinases consisting of pyruvate dehydrogenase kinase isoforms 1-4 (PDKs 1-4) and branched-chain alpha-keto acid dehydrogenase kinase (BDK) are negative metabolic switches that control glucose and branched-chain amino acids (BCAA) oxidation, respectively. These mitochondrial protein kinases deactivate pyruvate dehydrogenase complex (PDC) and the branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by reversible phosphorylation. Recent studies have established the PDK4 and BDK are up-regulated in animal models and patients with obesity and type 2 diabetes (T2D). The inhibition of PDKs and BDK results in enhanced glucose and BCAA oxidation. The results suggest a therapeutic approach to mitigate impaired glucose and BCAA disposal in obesity and T2D. However, to date there have been no potent selective PDK and BDK inhibitors for treatment of these metabolic disorders. The PI's laboratory recently employed structure-based design and high-throughput screening to develop robust highly selective liver-targeting inhibitors for both PDKs and BDK, which were shown to improve glucose tolerance and reduce hepatic steatosis in animal models. In this application, we will test the central hypothesis that mitochondrial PDKs and BDK are novel drug targets for pharmacological approach to obesity and Type 2 diabetes. Specific Aims are: 1) To provide molecular mechanisms for reduced hepatic steatosis in PDK inhibitor-treated mouse models and to establish liver as a target organ for treatment of obesity and T2D; and 2) To decipher the cause-effect relationship between increased BCAA levels and insulin resistance by enhancing BCKDC flux with BDK inhibitors. The successful outcome of this investigation will provide a framework for developing a new pharmacological approach to the treatment of obesity and T2D.